Antifungal agents

ABSTRACT

This invention relates to methods for inhibiting fungal growth employing compounds of formula (I) ##STR1##

BACKGROUND OF THE INVENTION

This is a continuation-in-part of copending patent application Ser. No.07/670,638 filed Mar. 18, 1991, now abandoned.

DESCRIPTION OF THE INVENTION

The present invention is directed to novel compounds of structuralformula (I) which are useful as inhibitors of fungal growth. ##STR2##wherein Z is selected from the group consisting of: a) H;

b) C₁₋₅ alkyl;

c) C₁₋₅ alkyl substituted with a member of the group consisting of:

i) phenyl,

ii) phenyl substituted with methyl, methoxy, halogen (Cl, Br, F, I) orhydroxy; or

a pharmaceutically acceptable salt of a compound of formula (I).

In one embodiment of the present invention are those compounds offormula (I) wherein the relative configuration of the tricyclic ring isas shown below: ##STR3## Throughout this specification and claims wherestereochemistry is described for the tricyclic ring the configurationimplied is relative. The actual configuration may be as shown or that ofits enantiomer.

Further illustrating this embodiment are those compounds of structuralformula (I) wherein the relative configuration at the 11-position is asshown below: ##STR4##

In one class of this embodiment are those compounds of structure (I)wherein the relative configuration at the 6-position is as shown below:##STR5##

Exemplifying this class is the compound wherein Z is hydrogen or apharmaceutically acceptable salt thereof. The compound wherein Z ishydrogen is hereafter referred to as Compound A.

Further illustrating this class are those compounds in which Z is C₁₋₅alkyl or C₁₋₅ alkyl substituted with phenyl or substituted phenylwherein the substituent is methyl, methoxy, halogen or hydroxy. In aspecific illustration Z is methyl. This compound is hereafter referredto as Compound B.

The compounds of formula (I) are prepared in an aerobic fermentationprocedure employing a novel culture, MF2664 identified as Aspergillusversicolor (Fungi, Class Hyphomycetes). Although the use of thisorganism is specifically described herein, other organisms of the genusAspergillus specifically including mutants of the above describedorganism are also capable of producing compounds of this invention andare included within this invention.

The culture MF2664 has been deposited, under the Budapest Treaty, withthe American Type Culture Collection at 12301 Parklawn Drive, Rockville,Md. 20852 as ATCC 74035. The culture MF2664, identified as a strain ofAspergillus versicolor exhibits the following morphological features:

Colonies attaining a diameter of 15 mm on yeast-malt extract agar(Difco) at 20° C. 12/12 hr light/dark in 7 days; no growth on the samemedium at 37° C. in 7 days; attaining a diameter of 10 mm on cornmealagar (Difco) at 20° C.; no growth on cornmeal agar at 37° C. Onyeast-malt extract agar, colonies up to 1 mm deep, moderately raised,velutinous to slightly floccose, dull, margin submerged, entire, hyalineto pale greenish yellow, Pale Chalcedony Yellow, Light Chalcedony Yellow(capitalized color names from Ridgway, R. 1912. Color Standards andNomenclature, Washington, D.C.) at the margin, soon dull yellowish greento green, Malachite Green, Light Bice Green, Forest Green, often withmottled appearance due to uneven pigment development in conidial heads,reverse dull yellow gray at the margin, but soon grayish to reddishbrown, Isabella Color, Vinaceous Brown, Dark Vinaceous Brown, Hay'sBrown, Seal Brown, with similar reddish brown, diffusible pigment exudedfrom the edges of the colony a few mm into the agar.

Conidiophores arising from a foot cell, 170-500 μm tall, 4.5-7 μm wide,straight to slightly curved or flexuous, sometimes with irregularconstrictions, often slightly constricted just below the vesicle,thick-walled, with walls 0.5-1 μm thick, smooth, hyaline to pale gray.Conidial heads 60-180 μm in diameter, biseriate with groups ofconidiogenous cells arising from metulae, at first loosely columnar butconidial chains soon splitting to become radiate, a first pale yellow,but soon becoming dull yellowish green to green, often with basalportion remaining pale yellow at maturity, rarely remaining entirelypale yellow at maturity. Conidiogenous cells phialidic, arising frommetulae in groups of 1-4, usually 3, 4-7×2-3.5 μm, cylindrical toampulliform, with distal end tapered to broadly truncate. Metulaebroadly cylindrical to clavate, 4.5-7.5×3-4.5 μm. Vesicles 10-15 μm indiameter, subglobose to pyriform, with metulae covering the upper60-80%. Conidia 2-4.5 μm in diameter, globose to subglobose, minutelyechinulate or roughened, hyaline to grayish green in KOH, adhering inchains by colorless connectives. Hyphae septate, smooth, highlybranched, often with intercalary or terminal, globose to subglobosecells, up to 10 μm in diameter, sometimes directly giving rise to singleor small groups of 3-4 phialidic conidiogenous cells. Huelle cells,sclerotia, and cleistothecia absent.

Aspergillus versicolor is a common and widely distributed organism,being reported from arctic to tropical regions from diverse soil types,detritus, plant litter, textiles, food products, animal dung, and livinganimals. Strain MF2664 can be assigned to Raper and Fennell's A.versicolor group (K. B. Raper & D. I. Fennell, The Genus Aspergillus,1965, Williams & Wilkins, Baltimore) based on the combination of:biseriate conidial heads; relatively long, colorless conidiophores;predominantly dull green pigmentation of the conidia; and lack of Huellecells and sclerotia. Within the A. versicolor group, MF2664 can bedistinguished from the other species by the smooth conidiophore walls,relatively small, echinulate conidia, and the dull green conidial heads.According to Raper and Fennell's monograph, A. versicolor is highlyvariable with regard to colony pigmentation, as the specific epithetindicates. The dull yellowish green to green conidial heads of MF2664,along with the dull vinaceous brown reverse and diffusible pigmentsappear to occupy a central position among the numerous color variantsdescribed within this broadly circumscribed species. Likewise, thecharacteristics of MF2664 agree well with the more modern and succinctspecies concept of A. versicolor presented by Domsch et al., 1980(Domsch, K. H., W. Gams, T. Anderson, 1980. Compendium of soil fungi.Academic Press, London).

Compounds of this invention can be obtained by culturing an above-notedmicroorganism on a solid or in an aqueous nutrient medium containingsources of assimilable carbon and nitrogen, preferably under aerobicconditions. Nutrient media may also contain mineral salts and defoamingagents.

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, glycerin, starch, dextrin, and the like. Other sourceswhich may be included are maltose, mannose, sucrose, and the like. Inaddition, complex nutrient sources such as oat flour, corn meal, millet,corn and the like may supply utilizable carbon. The exact quantity ofthe carbon source which is used in the medium will depend, in part, uponthe other ingredients in the medium, but is usually found in an amountranging between 0.5 and 15 percent by weight. These carbon sources canbe used individually in a given medium or several sources in combinationin the same medium.

The preferred sources of nitrogen are amino acids such as glycine,methionine, proline, threonine and the like, as well as complex sourcessuch as hydrolyzed proteins, yeast extracts (hydrolysates, autolysates),dried yeast, tomato paste, soybean meal, peptone, corn steep liquor,distillers solubles, malt extracts and the like. Inorganic nitrogensources such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate,ammonium phosphate, etc.) can also be used, as well as organic sourcessuch as urea. The various sources of nitrogen can be used alone or incombination in amounts ranging between 0.2 to 90 percent by weight ofthe medium.

The carbon and nitrogen sources are generally employed in combination,but need not be in pure form. Less pure materials which contain tracesof growth factors, vitamins, and mineral nutrients may also be used.Mineral salts may also be added to the medium such as (but not limitedto) calcium carbonate, sodium or potassium phosphate, sodium orpotassium chloride, magnesium salts, copper salts, cobalt salts and thelike. Also included are trace metals such as manganese, iron,molybdenum, zinc, and the like. In addition, if necessary, a defoamingagent such as polyethylene glycol or silicone may be added, especiallyif the culture medium foams seriously.

The preferred process for production of compounds of this inventionconsists of inoculating spores or mycelia of the producing organism intoa suitable medium and then cultivating under aerobic condition.

The fermentation procedure generally is to first inoculate a preservedsource of culture into a nutrient seed medium and to obtain, sometimesthrough a two step process, growth of the organisms which serve as seedsin the production of the active compounds. After inoculation, the flasksare incubated with agitation at temperature ranging from 20° to 30° C.,preferably 25° to 28° C. Agitation rates may range up to 400 rpm,preferably 140 to 220 rpm. Seed flasks are incubated over a period of 2to 10 days, preferably 2 to 4 days. When growth is plentiful, usually 2to 4 days, the culture may be used to inoculate production mediumflasks. A second stage seed growth may be employed, particularly whengoing into larger vessels. When this is done, a portion of the culturegrowth is used to inoculate a second seed flask incubated under similarconditions but employing shorter time.

After inoculation, the fermentation production medium is incubated for 3to 30 days, preferably 4 to 14 days, with or without agitation(depending on whether liquid or solid fermentation media are employed).The fermentation is conducted aerobically at temperatures ranging from20° to 40° C. If used, agitation may be at a rate to 400 rpm. To obtainoptimum results, the temperatures are in the range of 20° to 28° C.,most preferably 24° to 26° C. The pH of the nutrient medium suitable forproducing the active compounds is in the range of 3.5 to 8.5, mostpreferably 5.0 to 7.5. After the appropriate period for production ofthe desired compound, fermentation flasks are harvested and the activecompound isolated.

A polar solvent such as an ester, ketone or alcohol may be used toextract a compound of this invention from the solid fermentation medium.A mixture of an ester or ketone and an alcoholic solvent may also beemployed.

The mixture is vigorously stirred, filtered, and the filtrateconcentrated under reduced pressure. Methanol and water are added to theconcentrate, which is then extracted with a water immiscible solvent.The aqueous methanolic layer is removed and evaporated to dryness. Theresidue is then subjected to several separation steps such as adsorptionand chromatography. Fractions are collected and combined after eachseparation step based on biological assay and/or high performance liquidchromatography (HPLC) analysis.

The preferred solvent for extraction of the solid fermentation is ethylacetate. After concentration, the preferred partitioning solvent is a6:4 mixture of hexane and isopropyl acetate.

The chromatographic separations may be carried out using conventionalcolumn chromatography. Silica gel is the preferred initial adsorbent.When silica is the adsorbent, an ester/chlorohydrocarbon/organic acidmixture such as ethyl acetate/methylene chloride/acetic acid is usefulas an eluant. For reversed phase chromatography the preferred adsorbentis a C-18 bonded phase silica gel. The preferred eluant for reversedphase chromatography is a mixture of acetonitrile and water buffered toa neutral pH with a buffer such as 0.1M phosphate. It is preferable todo the isolation in the absence of light.

The pharmaceutically acceptable salts of the compounds of this inventioninclude those formed from cations such as sodium, potassium, aluminum,calcium, lithium, magnesium, zinc, and from bases such as ammonia,ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine,choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine,procaine, N-benzylphenethylamine, diethylamine, piperazine,tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide.

The present compounds exhibit broad spectrum antifungal activity asdemonstrated by a fungal inhibitory spectrum profile (FISP). The FISPassay employs disc diffusion susceptiblity testing methods to generate aspectrum of activity against a panel of representative yeasts,filamentous fungi and bacteria.

The filamentous fungi used in the assay are Penicillium sp. Aspergillusniger, Trichoderma sp., Phoma sp., Trichoderma lignorum, Fusariumoxysporum, Ustilago maydis, Ceratocystis ulmi, Alternaria soloni,Verticillium serrae, Botrytis allii, Scopulariopsis communis,Cephalosporium sp., Cercospora beticola, Rhizomucor miehei, Aspergillusflavus and Aspergillus fumigatus.

The yeasts employed in the assay are Saccharomyces cerevisiae, Candidaalbicans, Candida tropicalis, Candida rugosa, Brettanomycesbruxellensis, Torulospora hansenii, Candida guilliermondi, Candidapseudotropicalis, Torulopsis glabrata, Cryptococcus albidus,Cryptococcus laurentii and Kluyveromyces fragilis.

The bacteria employed in the assay are Streptomyces sp., Acholeplasmalaidlawii and Bacillus subtilis.

For carrying out the assay, seeded assay plates are prepared in thefollowing manner according to the type of assay strain.

Inocula for filamentous fungi are prepared by scraping the surface ofstock plates maintained on potato dextrose agar with a moistened steriledacron swab. The spores and mycelia are then suspended in 10 millilitersof sterile potato dextrose broth and adjusted to 65 percent transmissionat 660 nm.

Inocula for yeasts and bacterial strains are prepared from overnightbroth cultures which are then diluted into potato dextrose agar to afinal concentration of either 40 percent or 70 percent transmission at660 nm.

When the organism is Acholephasma laidlawii, it is suspended in brainheart infusion broth supplemented with 2.5 percent yeast extract and 20percent heat inactivated horse serum, and the broth adjusted to a finalconcentration of 60 percent transmission at 660 nm.

For three strains of Candida albicans and one strain of Saccharomycescerevisiae, sterile saline is employed in place of potato dextrosebroth.

Assay plates for the yeasts and filamentous fungi are prepared bydiluting the inoculum into appropriate molten agar medium (45° C.) toobtain a final concentration of 4 percent.

Seeded agar for Bacillus subtilis are prepared from a commerciallyavailable spore suspension which is diluted directly into molten agar(45° C.) to obtain a final concentration of 0.1 percent.

The seeded agar media thus prepared are dispensed into petri dishes forassays (11 milliliters per dish).

The samples to be tested for production of antifungal agent are appliedto 6.2 mm. filter paper discs (25 microliter/disc) and air dried at 24°C. When the sample to be tested is crude broth, it may be centrifugedprior to application. The discs bearing the material to be tested arethen applied employing sterile conditions to the seeded assay plates.The assay plates are then incubated at either 28° C. or 37° C. for 24hours. In one of the assays for Bacillus subtilis, the sample is appliedto a disc previously impregnated with 4M potassium chloride at 25μL/disc and then air dried.

Following incubation, the inhibition zones are measured and recorded.The measurements are made from the extreme edge where growth differsfrom the background zone. The zones are noted as to appearance as fuzzyedge and clear center, hazy throughout, slightly hazy, very hazy orringed.

The above tests as noted in the FISP profile of Table I demonstrate abroad spectrum of antifungal activity rendering the compounds useful ina wide variety of applications.

The broad spectrum antifungal activity of the present compounds wasfurther determined by broth dilution methods. The compounds areparticularly active towards filamentous fungi and yeasts includingCandida albicans and Crypt. neoformans. The sensitivity of filamentousfungi and yeasts was determined using inhibitor dilution assays inmicrotiter format. A standardized spore suspension for testing thefilamentous fungi was prepared by inoculating sterile distilled waterwith spores such that 1.5-7.5×10³ colony forming units were added perwell. The microtiter wells were filled with 75 μL of yeast nitrogen basecontaining 1% Dextrose medium (YNB/D) containing compound. Fifty μL ofinoculated YNB/D medium was then added to the wells.

The sensitivity of yeasts was determined by inoculating steriledistilled water with aliquots of a 4-hour yeast culture grown in YeastMorphology (YM) media at 35° C. and diluting in YNB/D to yield a finalconcentration of 1.5-7.5×10³ colony forming units/well. Seventy-five μLof inoculated media was added per well. To test the sensitivity ofyeast, compound was solubilized in 10 percent aqueous DMSO at 2.56mg/mL. The compound was diluted serially in YNB/D from 128 to 0.06μg/mL. The wells were filled with 75 μL of drug containing media. Theminimum inhibitory concentration (MIC in μg/mL) is defined as the lowestconcentration to prevent growth after an incubation for 48-72 hours, at35° C. for the filamentous fungi and 24 to 48 hours, at 35° C. for theyeasts. The minimum fungicidal concentration for yeasts in μg/mL isdefined as the lowest concentration of drug that totally preventedgrowth or permitted growth of no more than three colonies from a sampleremoved from each well and plated on Sabouraud agar media.Representative of the antifungal activity are the minimum inhibitoryconcentration and minimum fungicidal concentration data shown below inTable II:

                  TABLE I                                                         ______________________________________                                        FISP Profile of                                                               Compound A (CH.sub.2 Cl.sub.2, 250 μg/mL)                                  Strain               Zone                                                     ______________________________________                                        Filamentous Fungi                                                             Alternaria solani    26F                                                      Aspergillus flavus   26H                                                      Aspergillus fumigatus                                                                              24S                                                      Aspergillus niger    0                                                        Aspergillus niger    13H                                                      Botrytis allii       20S                                                      Cephalosporium sp.   17F                                                      Ceratocystis ulmi    10V                                                      Cercospora beticola  28S                                                      Fusarium oxysporum   14S                                                      Penicillium sp.      12H                                                      Penicillium sp.      12H                                                      Penicillium sp.      11H                                                      Phoma sp.            31S                                                      Rhizomucor miehei    23H                                                      Scopulariopsis communis                                                                            15F                                                      Trichoderma lignorum 31F                                                      Trichoderma sp.      33S                                                      Ustilago maydis      12V                                                      Verticillium serrae  35S                                                      Bacteria                                                                      Streptomyces sp.     8V                                                       Acholeplasma laidlawii                                                        Bacillus subtilis    0                                                        Bacillus subtilis - KCl                                                                            0                                                        Yeasts                                                                        Brettanomyces bruxellensis                                                                         32S                                                      Candida albicans     0                                                        Candida albicans - YNB                                                                             23V                                                      Candida albicans     30S                                                      Candida albicans     28S                                                      Candida albicans     24S                                                      Candida albicans     31S                                                      Candida albicans - YNB/GLU                                                                         34H                                                      Candida albicans - YNB/NAG                                                                         35H                                                      Candida guilliermondii                                                                             0                                                        Candida pseudotropicalis                                                                           14V                                                      Candida rugosa       30S                                                      Candida tropicalis   35S                                                       Cryptococcus albidus                                                                              11V                                                      Cryptococcus laurentii                                                                             13H                                                      Cryptococcus laurentii                                                                             13V                                                      Cryptococcus laurentii                                                                             11H                                                      Kluyveromyces fragilis                                                                             12V                                                      Saccharomyces cerevisiae                                                                           0                                                        Saccharomyces cerevisiae                                                                           12H                                                      Saccharomyces cerevisiae - YNB                                                                     16H                                                      Torulopsis glabrata  0                                                        Torulospora hansenii 20H                                                      ______________________________________                                    

FISP data is reported as the diameter (in mm) of the zone of inhibitionof growth in agar diffusion assays. The zone is determined by measuringgrowth that differs from the background lawn. A qualifier is also addedto describe the zone quality as listed below: no qualifer: clear zonethroughout, sharp edges

F: clear zone, fuzzy edges

S: slightly hazy zone (some growth observed throughout zone)

H: hazy zone (considerable growth observed throughout zone)

V: very hazy zone (zone of inhibition is barely discernable)

R: zone displays a ring of resistant growth

                  TABLE II                                                        ______________________________________                                        Minimum Inhibitory and Fungicidal                                             Concentration of Compound A in the Microdilution                              Broth Assay (MIC/MFC μg/mL)                                                Fungus             MIC      MFC                                               ______________________________________                                        Candida albicans                                                              MY 1055            ≦0.06                                                                           ≦0.06                                      MY 1028            ≦0.06                                                                           ≦0.06                                      Candida tropicalis                                                            MY 1012            ≦0.06                                                                            0.50                                             Candida parapsilosis                                                          MY 1010             0.25     0.25                                             Candida guilliermondii                                                        MY 1019            >128     >128                                              Saccharomyces cerevisiae                                                      MY 1976            >128     >128                                              Cryptococcus neoformans                                                       MY 1051            32       32                                                MY 2061            128      64                                                MY 2062            64       16                                                MY 1146            128      64                                                Aspergillus fumigatus                                                         MF 4839            64                                                         T. mentagrophytes                                                             MF 4864            32                                                         ______________________________________                                    

Thus the present invention is also directed to a method of treatingfungus infections which comprises the administration to an organism inneed of such treatment a nontoxic therapeutically effective amount of acompound represented by the structural formula (I) and pharmaceuticallyacceptable salts thereof. Based on the above MIC data it is determinedthat generally from 1 to 10 mg/kg should be employed as a unit dosage inan antifungal treatment.

The compounds of this invention are adaptable to being utilized invarious applications of antifungal compositions. In such use, compoundsmay be admixed with a biologically inert carrier, generally with the aidof a surface active dispersing agent, the nature of which would varydepending on whether the use is for the control of pathogens infectingmammals such as man, or birds or reptiles, or for control of fungi inagriculture such as in soil or plant parts, or for the control of fungiin inanimate objects.

In compositions for medical applications, the compounds may be admixedwith a pharmaceutically acceptable carrier, the nature of which willvary depending on whether the composition is to be topical, parenteralor oral.

If said application is to be topical, the drug may be formulated inconventional creams and ointments such as white petroleum, anhydrouslanolin, cetyl alcohol, cold cream, glyceryl monostearate, rose waterand the like.

For parenteral applications, the compounds may be formulated inconventional parenteral solutions such as 0.85 percent sodium chlorideor 5 percent dextrose in water, or other pharmaceutically acceptablecompositions.

Compositions for oral administration may be prepared by intimatelymixing the component drugs with any of the usual pharmaceutical media,including, for liquid preparations, liquid carriers such as water,glycols, oils, alcohols, and the like; and for solid preparations suchas capsules and tablets, solid carriers such as starches, sugars,kaolin, ethyl cellulose, surface active dispersing agents, generallywith lubricant such as calcium stearate, together with binders,disintegrating agents and the like.

These compositions are then administered in amounts sufficient to obtainthe desired antifungal effect. For medical application, the methodcomprises administering to a subject in need of treatment atherapeutically effective antifungal amount of a compound of Formula I.The appropriate doses will vary depending on age, severity, body weightand other conditions. For topical application the compositions areapplied directly to the area where control is desired. For internaladministration, the composition may be applied by injection or may beadministered orally.

For non-medical application, the product of the present invention,either singly or as a mixture, may be employed in compositions in aninert-carrier which includes finely divided dry or liquid diluents,extenders, fillers, conditioners and excipients, including variousclays, diatomaceous earth, talc, and the like, or water and variousorganic liquids such a lower alkanols, for example ethanol andisopropanol, or kerosene, benzene, toluene and other petroleumdistillate fractions or mixtures thereof.

These compositions may be employed by applying to the surface of orincorporating in the medium to be protected. For the control of riceblast, tomato late blight, tomato early blight, wheat leaf rust, beanpowdery mildew and tomato Fusarium wilt, the compositions may be applieddirectly to the plant in topical application or administered to the soilfor systemic application. The method comprises administering to theaffected plant, soil or medium to be protected an antifungally effectiveamount of the compound of Formula I.

The following examples illustrate the preparation of the compounds offormula (I) and their incorporation into pharmaceutical compositionsand, as such, are not to be considered as limiting the invention setforth in the claims appended hereto.

The composition of media employed in the following Examples are listedbelow:

    ______________________________________                                        YME Seed Medium                                                                      Component                                                                              (g/L)                                                         ______________________________________                                               Yeast Extract                                                                          4.0                                                                  Malt Extract                                                                           10.0                                                                 Dextrose 4.0                                                           ______________________________________                                    

The medium was prepared with distilled water and the pH adjusted to 7.0prior to sterilization and sterilized at 121° C. for 20 minutes. Themedium was dispensed at 54 mL/250 mL plain Erlenmeyer flask. Cottonclosures were used.

NPF-2 PRODUCTION MEDIUM

1. Solid phase:

Vermiculite: 1250 cc/4 liter roller jar. Latex closure. The vermiculiteportion of the medium was sterilized separately for 60 minutes at 121°C.

2. Liquid phase:

    ______________________________________                                        Component         (g/L)                                                       ______________________________________                                        Dextrose          150.0                                                       Urea              4.0                                                         NZ amine Type A   4.0                                                         K.sub.2 HPO.sub.4 0.5                                                         MgSO.sub.4.7H.sub.2 O                                                                            0.25                                                       KCl                0.25                                                       ZnSO.sub.4.7H.sub.2 O                                                                           0.9                                                         CaCO.sub.3        16.5                                                        ______________________________________                                    

The medium was prepared with distilled water (no pH adjustment). Themedium was dispensed at 425 mL/1 L Erlenmeyer flask. Cotton closureswere used, and the medium was sterilized at 121° C. for 15 minutes.

    ______________________________________                                        KF Seed Medium    Trace Elements #2                                           Component    g/L      Component      g/L                                      ______________________________________                                        Glucose      10.0     FeSO.sub.4.7H.sub.2 O                                                                        1.0                                      Corn Steep Liquor                                                                           5.0     MnSO.sub.4.4H.sub.2 O                                                                        1.0                                      Tomato Paste 40.0     CuCl.sub.2.2H.sub.2 O                                                                        0.025                                    Oat Flour    10.0     CaCl.sub.2     0.1                                      Trace Elements #2                                                                          10.0 mL  H.sub.3 BO.sub.3                                                                             0.056                                    Adjust pH = 6.0       (NH.sub.4).sub.6 MoO.sub.2.4H.sub.2 O                                                        0.019                                                          ZnSO.sub.4.7H.sub.2 O                                                                        0.2                                                            HCl (Conc)     5.0 mL                                   ______________________________________                                    

EXAMPLE 1 Preparation of Compound A A. Culture and Fermentation

i) Preparation of FVM

A lyophilized culture of MF2664 obtained from the Merck CultureCollection was used to prepare frozen vegetative mycelia (FVM) byaseptically transferring the entire contents of the lyophilizedpreparation into a seed flask (YME, 54 mL/250 mL flask) and incubated at25° C., 220 rpm for 4 days. The FVM were frozen at -75° C. in 10-15%glycerol. Secondary FVM were prepared from a primary FVM in YME seedmedium and frozen as heretofore described.

ii) Seed Culture

Seed cultures of MF2664 Aspergillus versicolor, were inoculated with 1.0mL of the FVM and grown on a gyratory shaker (220 rpm) for 4 days in YMEseed (54 mL/250 mL flask) at 25° C. Approximately 10 small sterileceramic balls and 5 small, sterile ceramic cylinders were added to theflask, which was then incubated on a gyratory shaker for about 2 hours.

iii) Production (one roller jar)

A portion of the seed culture (18 mL) was used to inoculate the liquidportion of a production medium (liquid phase, 425 mL/1 L flask). Thismixture was added to a 4 liter vermiculite-containing roller jar (solidphase). The roller jar production vessel was incubated on a rollermachine at 25° C. for 13 days.

B. Isolation of Compound A

The contents of fourteen roller bottles of the solid fermentation eachprepared as described above in part (A) were combined and extractedtwice with a total of 18 L of ethyl acetate. The mixture was then vacuumfiltered through Whatman #1 filter paper to remove insolubles. An 830 mgfraction was concentrated by rotary evaporation and subsequentlyreconstituted in 90% methanol/10% water. This 90% methanol solution waspartitioned (1:1) with 6:4 hexane/isopropyl acetate. The methanol/waterlayer was lyophilized and reconstituted in 4 mL of ethylacetate/methylene chloride (1:1)+1% acetic acid. This comprised the feedfor an 80 mL silica column.

Activity was eluted from the 80 mL silica column using a step gradientbeginning with 7:3 methylene chloride/ethyl acetate+1% acetic acid andending with methanol. The majority of the activity eluted in fractions8-18, which represented 200 mL of the 1:1 methylene chloride/ethylacetate+1% acetic acid elution. These fractions were pooled,concentrated to dryness and weighed. The pooled sample which weighed 100mg, was resuspended in 1.5 mL of methanol. A 0.5 mL aliquot (33 mg) waschromatographed on a Prep-RP-HPLC (Phenomenex ODS 30, 250×22.5, 30:70acetonitrile 0.01M phosphate buffer pH 7, flow=10 mL/min, UV=320 nm, 10mL/fraction. The compound A component eluted in fractions 23-26. Bufferwas removed from these active fractions by partitioning with one volumeof methylene chloride+1% acetic acid. The activity resided in themethylene chloride layer which after evaporation yielded Compound A.Compound A was found to be light sensitive but stable when isolated andworked with in the absence of light.

EXAMPLE 2 Alternate Culture and Production Procedure

A lyophilized culture of MF2664 from the Merck culture collection wasplaced into 50 mL of KF seed medium in a 250 mL baffled Erlenmeyerflask. This flask was shaken at 220 rpm (2 inch throw New Brunswickshaker) and at 27° C. for 5 days. At the end of this incubation period a2.5 mL portion of the culture broth was mixed with 0.5 mL of sterile 60%glycerol and portions of this suspension were frozen in six sealed glassvials at -80° C. until use. One of the vials containing MF2664 wasthawed to room temperature and was used to inoculate 50 mL of YME seedmedium contained in a 250 mL baffled Erlenmeyer flask. This flask wasincubated at 25° C., 220 RPM for 3 days. The resulting biomass wasaseptically macerated with 12 mm porcelain balls and 24 mL of the slurrywas placed into a 425 mL portion of production medium NPF2. Theproduction medium was shaken to disperse the biomass and was added to a110×535 mm roller culture bottle containing 1250 cc of sterilelarge-particle vermiculite. The roller culture bottle was shaken well todistribute the contents and was incubated on a roller assembly at 25°C., 75% relative humidity for 18 days.

EXAMPLE 3

As a specific embodiment of an oral composition of a compound of thisinvention, 200 mg of the compound from Example 1 is formulated withsufficient finely divided lactose to provide a total amount of 580 to590 mg to fill a size 0 hard gelatin capsule.

EXAMPLE 4 Preparation of an Ammonium Salt

A 0.1 mmol sample of the free acid of a compound of formula (I) isdissolved in 10 mL of ethyl acetate. The resulting solution is saturatedwith gaseous ammonia upon which the ammonium salt precipitates fromsolution.

EXAMPLE 5 Preparation of a Potassium Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) in10 mL of methanol is treated with an aqueous or methanolic solutioncontaining 0.1 mmol of potassium hydroxide. Evaporation of the solventaffords the potassium salt. In a similar fashion the sodium and lithiumsalts can be formed.

EXAMPLE 6 Preparation of a Calcium Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) in20 mL of 6:4 methanol/water is treated with an aqueous solution of 0.05mmol of calcium hydroxide. The solvents are evaporated to give thecorresponding calcium salt.

EXAMPLE 7 Preparation of an Ethylenediamine Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) in10 mL of methanol is treated with 0.05 mmol of ethylenediamine.Evaporation of the solvent affords the ethylenediamine salt.

The procedure can also be applied to the preparation of theN,N"-dibenzylethylenediamine salt.

EXAMPLE 8 Preparation of a Tris(hydroxymethyl)aminomethane Salt

To a solution of 0.1 mmol of the free acid of a compound of formula (I)in 10 mL of methanol is added about 0.1 mmoltris(hydroxymethyl)aminomethane dissolved in 10 mL of methanol.Evaporation of the solvent gives the titled salt. Similarly prepared arethe salts of L-ornithine, L-lysine, and N-methylglucamine.

EXAMPLE 9 Preparation of an L-arginine Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) in10 mL of 6:4 methanol/water is treated with an aqueous solution of 0.1mmol of L-arginine. Evaporation of the solvent affords the title salt.

Similarly prepared are the salts of L-ornithine, L-lysine andN-methylglucamine.

EXAMPLE 10 Preparation of a Compound B

One mg of Compound A in 250 μL of methylene chloride was reacted at lowtemperature with several drops of diazomethane. Seventy-five μg wasremoved and both samples were immediately concentrated to dryness toquench the reaction. Mass spectral analysis confirmed the presence ofthe Compound B in the 75 μg sample and ¹ H-NMR confirmed the presence ofCompound B in the remaining sample.

EXAMPLE 11 General Method for Preparation of an Ester

A solution of 2 mg of Compound A in 0.5 mL of acetonitrile is treated atroom temperature with 2 equivalents of DBU and 2 equivalents of MeI. Thereaction is diluted after two hours with 10 mL of dichloromethane andwashed successively with 10 mL of 0.1M phosphoric acid, 10 mL of water,10 mL of saturated sodium bicarbonate and 10 mL of water. After dryingover sodium sulfate the organic layer is concentrated and the residue ischromatographed on silica gel using mixtures of dichloromethane andethyl acetate to give the ester.

The method described above is also suitable for the preparation of otherester derivatives such as ethyl and other lower alkyl esters and benzyland substituted benzyl esters.

Mass Spectral Data

Mass spectra were acquired on Finnigan-MAT models MAT212 and TSQ70 massspectrometers. (MATZ212: Electron Impact (EI) mode at 90 eV. Exact massmeasurements were performed at high resolution (HR EI) usingperfluorokerosene (PFK) as an internal standard. TSQ70: EI mode at 70eV. Fast Atom Bombardment (FAB) mode employing negative ion detectionand ethanolamine as the matrix.)

NMR Spectra

All NMR spectra were acquired on a Varian Unity 500 spectrometeroperating at 499.843 MHz for proton measurements and 125.697 MHz for ¹³C measurement. Carbon-13 NMR studies and ¹ H NMR studies were performedon a 7 mM solution of Compound A in either CD₂ Cl₂ or a mixture of 85:15CD₃ CN/CD₂ Cl₂ at ambient temperature (20°+/-1° C.).

Physical Properties of the compounds of Structure I:

Compound A--the compound of structure I wherein Z is H.

Mass Spectral Data

This compound has the molecular weight 540 by FAB-MS ([M-H]⁻ =539). Themolecular formula C₃₁ H₄₀ O₈ was determined by HR EI of the [M-H₂ O]⁺ion: calculated 522.2618, found 522.2623.

¹³ C NMR Data in CD₂ Cl₂ Solution:

Chemical shifts for ¹³ C spectra recorded in CD₂ Cl₂ solution are givenin ppm relative to tetramethylsilane (TMS) at zero ppm using the solventpeak at 53.8 ppm as an internal standard: 12.3, 13.3, 14.8, 17.8, 21.0,23.3, 27.3, 35.6, 40.2, 44.1, 47.3, 53.3, 70.2, 78.2, 79.7, 91.9, 119.7,128.2, 128.3, 128.9, 129.1, 130.4, 130.6, 136.1, 137.0, 140.5, 142.8,145.7, 171.5, 175.6, 209.1 ppm.

¹³ C NMR Data in 85:15 CD₃ CN/CD₂ Cl₂ Solution:

Chemical shifts for ¹³ C spectra recorded in 85:15 CD₃ CN/CD₂ Cl₂solution are given in ppm relative to tetramethylsilane (TMS) at zeroppm using the solvent peak of the acetonitrile methyl group at 1.3 ppmas an internal standard: 11.9, 13.2, 14.8, 18.1, 21.1, 23.3, 27.6, 35.8,40.4, 44.9, 47.8, 54.2, 70.2, 78.0, 80.2, 92.0, 120.9, 128.5, 128.6,129.2, 129.3, 131.1, 131.5, 136.2, 137.2, 141.3, 141.6, 145.2, 168.0,175.4, 209.9 ppm.

¹ H NMR Spectrum (500 MHz) (CD₂ Cl₂)

See FIG. 1

UV (MeOH) μ_(max) is at 240 and 340 nm.

IR (as free acid: film on ZnSe) representative peaks 1726, 1709, 1610,1570, 1455, 1410 cm⁻¹.

Compound B--the mono-methyl ester of the compound of structure (I)wherein Z is methyl.

Mass Spectral Data

This compound has the molecular weight of 554 by LR EI analysis.

What is claimed is:
 1. A method for inhibiting fungal growth comprisingapplying to the area where growth is to be controlled an antifungallyeffective amount of a compound of formula (I): ##STR6## wherein Z isselected from the group consisting of: a) H;b) C₁₋₅ alkyl; and c) C₁₋₅alkyl substituted with a member of the group consisting of:i) phenyl,and ii) phenyl substituted with methyl, methoxy, halogen or hydroxy; orapharmaceutically acceptable salt of a compound of formula (I).
 2. Amethod for inhibiting fungal growth in a living organism in need of suchtreatment comprising the oral, parenteral or systemic administration ofan effective amount of a compound of claim 1 to said living organism. 3.A method of claim 2 wherein the living organism is a mammal.
 4. A methodof claim 2 wherein the living organism is a bird.
 5. A method of claim 2wherein the living organism is a plant.
 6. A method of claim 2 whereinthe compound is selected from the group consisting of: ##STR7##